摘要:
Objective To explore the role of nuclear factor (NF)-κB signal pathway in the process of hepatic veno-occlusive disease (HVOD) after allogeneic bone marrow transplantation (allo-BMT) in mice.Methods A total of 50 male BALB/c(H-2Kd)mice aged 8-10 weeks were included in this study.First,all mice were pretreated with lethal dose of total body irradiation (TBI).Then according to different transplant program,these mice were divided into bone marrow transplantutaion (BMT) group (n=20) and bone marrow joint endothelial progenitor cells (EPC) transplantion group (EPC group) (n =20).The TBI control group (n=5) and normal control group (n=5) were also set up.Liver function and the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in serum were detected on day 7,15,33 after transplantation,and the histological changes of hepatocytes were evaluated by hematoxylin-eosin (HE) staining and Masson staining.Pathological changes in the liver of mice were observed by microscope.Then the activation of the NF-κB signal pathway was detected by quantitative polymerase chain reaction and Western blot.Results ①After the transplant,AST,ALT and TBIL levels in EPC group recipient mice were significantly lower,compared with BMT group.The differences between BMT and EPC group were statistically significant (P < 0.05).② The concentration of TNF-α in EPC group was (138.73 ± 10.42) pg/mL,which was higher than (95.31 ±19.09) pg/mL in BMT group,on the 7th days after transplantation,but concentration of IL-6 in EPC group [(169.50 ±10.21)] pg/mL was lower than that in BMT group [(95.31±19.09) pg/mL].The differences between BMT and EPC group were statistically significant (t=6.907,-6.372; P<0.05).On the 15th day after transplantation,the concentration of IL-6 in EPC group was significantly elevated to (201.49± 14.97) pg/mL,which was higher than (151.51 ±26.43) pg/mL in the BMT group,the difference between the two groups was statistically significant (t 5.51,P<0.05).③On the 33th day after transplantation,the transforming growth factor (TG-F)-β,CXCL2,CCL2 and CCR2 levels of EPC group were significantly lower than BMT group,these indicators between BMT and ECP group were statistically significant differences (P<0.05).④The expression of IKKβ in NF-κB signal pathway peaked on the 15th day.The activation of IKKβ,phosphorylation of Iκβα and activation of P65 were consistent with the expression of IKKβ.Conclusions Activation of NF-κB signal pathway may promote the repair of damaged hepatocytes in early stage of allo-BMT complicating HVOD mice,but NF-κB signal pathway sustained activation may promote the formation of liver fibrosis after the restore of impaired cells.%目的 探讨核因子(NF)-κB信号通路在异基因骨髓移植(allo-BMT)后并发肝静脉闭塞病(H VOD)中的作用.方法 选择50只8~10周龄雄性BALB/c(H-2Kd)小鼠作为研究对象.小鼠经致死剂量全身照射(TBI)预处理后,根据移植方案不同,将其分为骨髓移植(BMT)组(n=20)和骨髓联合内皮祖细胞(EPC)移植组(EPC组)(n=20);另设TBI对照组(n=5)和正常对照组(n=5)(实验过程中对动物的处置符合动物伦理学标准).于移植后7d、15d和33 d,检测移植后受鼠肝功能及血清肿瘤坏死因子(TNF)-α及白细胞介素(IL)-6水平.受鼠肝脏组织经苏木精-伊红(HE)染色法及Masson染色法染色后,光镜下观察小鼠肝脏的病理改变.荧光定量聚合酶链反应及Western免疫印迹法(WB)法检测受鼠肝细胞内NF-κB信号通路的活化情况.结果 ①移植后,EPC组受鼠与BMT组相比,天冬氨酸转氨酶、丙氨酸转氨酶和总胆红素水平明显降低,两组比较,差异有统计学意义(P<0.05).②移植后第7天,EPC组受鼠TNF-α表达量为(138.73±10.42)pg/mL,显著高于BMT组的(95.31±19.09) pg/mL,两组相比,差异有统计学意义(t=6.907,P<0.05),而IL-6的表达量为(169.50±10.21) pg/mL,低于BMT组的(215.01±22.13)pg/mL,两组比较,差异有统计学意义(t=-6.372,P<0.05);第15天EPC组受鼠的IL-6水平升高至(201.49±14.97) pg/mL,明显高于BMT组的(151.51±26.43) pg/mL,两组比较,差异有统计学意义(t=5.51,P<0.05).③移植后第33天,EPC组受鼠转化生长因子(TGF)-β、CXCL2、CCL2和CCR2水平均较BMT组明显降低,两组上述各项指标比较,差异均有统计学意义(P<0.05).④移植后小鼠肝细胞内NF-κB信号通路相关分子IKKβ于移植后第15天达峰值.IKKβ的活化、IκBα的磷酸化及P65的活化水平与IKKβ的表达水平一致.结论 allo-BMT并发HVOD受鼠早期肝细胞内NF-κB信号通路的活化,可促进损伤肝细胞的修复,但在肝细胞修复后,NF-κB信号通路的持续活化可促进肝纤维化.